- ISH (1) (remove)
- In situ studies of sugar metabolism in Ricinus communis L. and Saccharum officinarum L. (2004)
- In order to find the sucrose efflux transporter of the endosperm of Ricinus communis L., the yeast complementation selection method was used, but it was unsuccessful. Mutation was occurred on the nSC4+ plasmid during the selection. Maybe the stress of the yeast cells was too strong and then induced the mutation in the yeast cells. Using a weak promoter and reducing the copy number of the plasmid may avoid the mutation occur during the selection. The endosperm of Ricinus communis L. stores lipid and converts it to sucrose for the growth of seedlings. Sucrose phosphate phosphatase gene, RcSPP1, was cloned from the endosperm of the germinating seedling of Ricinus communis L. The endosperm cells synthesize sucrose by using SPS and SPP rather than sucrose synthase. Northern blot analysis indicated that the RcSPP1 expression level of the germinating endosperm was very similar from day 2 to day 6. The expression of nsLTPc1 is cotyledon-specific. It is also confirmed by in situ hybridization. The results of nsLTPc1 in situ hybridization indicate that the expression of nsLTPc1 was a cell-specific. The expression of nsLTPc1 was found only in the lower side of the cotyledons of Ricinus communis L. The expression of RcSCR1 is found in the endosperm, hypocotyl and cotyledons of the Ricinus communis L. germinating seeds. By northern blot analysis of the RNA from different days old endosperm, it indicates that the RcSCR1 has a highest expression level at day 5. By in situ hybridization and immunolocalization, the results illustrate that the mRNA and protein can be found in the lower epidermis of cotyledons from day 2 to day 5. In the 6-day-old cotyledons, the mRNA and protein of RcSCR1 are predominantly found in palisade parenchyma cells, but they are also found in the lower epidermis of cotyledons. The results of in situ hybridization indicate that the transcript of RcSCR1 can be found in most of the endosperm cells. RcSCR1 can be found in the middle layer of the endosperm from day 2 to day 5, no transcript of RcSCR1 is found in the cell layers near the seed coat. On the day 6, no RcSCR1 transcript can be detected in the endosperm cells. It is suggest that the function of RcSCR1 protein is to retrieve the sucrose from apoplastic space to avoid sucrose escape. Compared to the amino acid sequence of known sucrose transporters, the putative sucrose transporter of Ricinus communis , RcSCR2, belongs to SUT4 subfamily. The transcript of RcSCR2 is found in the endosperm, hypocotyl and cotyledons of the Ricinus communis L. germinating seeds. The expression of RcSCR2 is very weak. The expression level of RcSCR2 cannot be detected by northern analysis. By quantitative real time RTPCR, it indicates that the RcSCR2 has a highest expression level at 3 day. By in situ hybridization, the results illustrate that the mRNA cannot be found in the endosperm, cotyledons and hypocotyl. The results of in situ RTPCR indicate that the transcript of RcSCR2 can be found in most of the endosperm cells. RcSCR2 can be found in the middle layer of the endosperm from day 2 to day 5, no transcript of RcSCR2 is found in the cell layers near the seed coat. On the day 6, no RcSCR2 transcript can be detected in the endosperm cells. Although RcSCR2 in yeast does not function properly, but it shares high homology to other SUT4 type transporters, so they may have the same function to take up sucrose into cells. It is suggest that the function of RcSCR2 protein is to retrieve the sucrose from the apoplastic space to avoid sucrose escape. How the expression of RcSCR1 and RcSCR2 is regulated in the endosperm is still unknown. Sugarcane is a very important food crop. Sugarcane yellow leaf virus leads to sugarcane yellow syndrome and reduces the sugar production. Starch accumulation was found in the virus-infected plants. Within the starch staining, the results indicate that starch is accumulated in bundle sheath cells and mesophyll cells of virus -infected plants, however, starch can be found only in the bundle-sheath cells of virus-free plants. The in situ hybridization study indicates that the expression of ADP-glucose pyrophosphorylase in the mesophyll cells of virus-infected plant is stronger than it in virus-free plants. The results of in situ hybridization of starch branching enzyme indicates that no significant difference between the virus-free-plants and virus-infected. The results are different to it of starch staining. The mechanisms are still unclear, more carbohydrate metabolism related genes must be studied.